a) Field of the Invention
The present invention relates to a method of observation in optical microscopy, specifically to that using a scanning fluorescence microscope, which achieves high spatial resolution to obtain an image with good quality by illuminating a dyed specimen with a plurality of light beams of different wavelengths.
b) Description of Related Art
Optical microscopes have a substantial history, in which various types of apparatuses have been developed and improved. On the other hand, progress in the background technology such as laser technology and electronic image technology in recent years has contributed to development of microscopes of nowadays with higher performance. In this situation, Japanese Patent Preliminary Publication No. Hei 8-248200, for example, proposes a high-performance microscope that makes it possible not only to control contrast of the image obtained but also to perform chemical analysis of the specimen. This type of microscope is explained below in spectroscopic terms.
FIG. 11 shows the electronic structure of valence electron orbits of a molecule contained in a specimen. According to the microscope disclosed by the above-mentioned Hei 8-248200, the double-resonance absorption process is used for observation of those absorption and fluorescence with respect to a selected molecule which result from specific optical transitions. The principle of the double resonance absorption process is explained below in reference to FIGS. 12-14.
First, electrons in valence electron orbits of a molecule in the ground state (hereinafter referred to as S.sub.0 state) shown in FIG. 11 are excited by light waves of a certain wavelength (.lambda..sub.1) to enter into the first-level excited state (hereinafter referred to as S.sub.1 state), as shown in FIG. 12. Second, the electrons are excited by light waves of a different wavelength (.lambda..sub.2) in a similar manner as shown in FIG. 13 to enter into the second-level excited state (hereinafter referred to as S.sub.2 state). The molecule in S.sub.2 state then emits fluorescence or phosphorescence to return to S.sub.0 state as shown in FIG. 14.
According to the method of microscopic observation where the double-resonance absorption process is utilized, the absorption process shown in FIGS. 12 and 13 and emission of fluorescence or phosphorescence shown in FIG. 14 are used for observation of an absorption image and a luminescence image.
In such a method, first, molecules in a specimen are excited by a resonance wavelength .lambda..sub.1 of laser light or the like to enter into S.sub.1 state shown in FIG. 12. The number of S.sub.1 -state molecules per unit volume (population) increases as the intensity of the light with which the specimen is irradiated increases. A linear absorption coefficient is given as the product of {absorption cross section per molecule} and {number of molecules per unit volume}. Therefore, in the molecular excited state shown in FIG. 13, the linear absorption coefficient for the light of resonance wavelength .lambda..sub.2, with which the molecules are irradiated in succession to the irradiation with the light of wavelength .lambda..sub.1, depends on the intensity of the light of the wavelength .lambda..sub.1. Accordingly, the linear absorption coefficient for the light of the wavelength .lambda..sub.2 is controllable by the intensity of the light of the wavelength .lambda..sub.1. With respect to a transmission image of the specimen formed with transmitted light of the wavelength .lambda..sub.2, it means that the contrast of the transmission image is fully controllable with amount of light of the wavelength .lambda..sub.1.
On the other hand, if decay from S.sub.2 state could occur as being accompanied by emission of fluorescence or phosphorescence as shown in FIG. 14, the luminous intensity is proportionally related with the number of S.sub.1 -state molecules. Accordingly, where the microscope is used as a fluorescence microscope, also, control of image contrast is possible. In addition, chemical analysis of the specimen also is possible in such application of the microscope.
The outermost shells, or valence electron orbits, which are shown in FIG. 11, possess energy levels peculiar to each molecule. Therefore, fluorescence caused by irradiation with light of wavelength .lambda..sub.1 varies with molecules irradiated. Similarly, when irradiated with the light of the wavelength .lambda..sub.2, a molecule emits fluorescence peculiar to it. If a specimen is irradiated with light of a single wavelength, as conventionally done, absorption image or fluorescence image of a certain molecule is observable to some degree. However, the chemical composition of the specimen cannot be accurately identified, because, in general, the wavelength ranges of the absorption bands of several molecules overlap one another.
In contrast, according to the method of microscopic observation utilizing the double-resonance absorption process, molecules to absorb or emit light are limited by two wavelengths .lambda..sub.1 and .lambda..sub.2, and thus accurate identification of chemical composition of the specimen is possible. Furthermore, when a valence electron is excited, light having a specific electric field vector in reference to a molecular axis is strongly absorbed. Therefore, if the absorption image or the fluorescence image is photographed upon directions of polarization of light waves of wavelengths .lambda..sub.1 and .lambda..sub.2 being controlled, orientations of individual molecules of one kind can be identified.
Furthermore, according to some recent proposals, a fluorescence microscope is able to have such a high spatial resolution as to overcome the diffraction limit by using the double-resonance absorption process. The principle of such a microscope is described in reference to FIG. 15.
FIG. 15 schematically shows the double-resonance absorption process concerning a certain molecule. The molecule in S.sub.0 state is excited by wavelength .lambda..sub.1 to enter into S.sub.1 state, and then is excited by wavelength .lambda..sub.2 to S.sub.2 state. As schematically illustrated, fluorescence from the S.sub.2 -state molecule is extremely weak.
A very interesting phenomenon can be expected with respect to molecules of a kind having the above-described optical property, as explained below in reference to FIG. 16. FIG. 16 introduces abscissa (direction of X) for presenting the spatial extension in the model of double-resonance absorption process similar to FIG. 15. The spatial region A.sub.1 represents a region irradiated with the wavelength .lambda..sub.2, while the spatial region A.sub.0 represents a region that is not irradiated with the wavelength .lambda..sub.2.
In the spatial region A.sub.0, a multitude of molecules in S.sub.1 state are generated by excitation by the light of the wavelength .lambda..sub.1, when fluorescence of a wavelength .lambda..sub.3 appears. In the spatial region A.sub.1, however, molecules with S.sub.1 state are excited to enter into S.sub.2 or much higher level state almost at the moment as produced, and thus cannot survive. This phenomenon is confirmed with respect to several kinds of molecules. Therefore, in the spatial region A.sub.1, fluorescence emission is thoroughly repressed, because fluorescence of wavelength .lambda..sub.3 is completely extinguished and decay from S.sub.2 state does not involve fluorescence. Consequently, fluorescence resides only in the spatial region A.sub.0.
This result is of great importance in microscopy. According to a conventional scanning laser microscope, a specimen is scanned by a microbeam, which is formed by focusing laser light. The irradiation region with the microbeam is determined by the diffraction limit, which is determined by the numerical aperture of the focusing lens and the wavelength of the laser light. In principle, spatial resolution cannot be free from the diffraction limit. However, if the beams of wavelengths .lambda..sub.1 and .lambda..sub.2 spatially overlap with each other in an appropriate manner, the fluorescence region is limited by irradiation with the wavelength .lambda..sub.2 as shown in FIG. 16. In this condition, the fluorescence region is narrower than the irradiation region with the wavelength .lambda..sub.1, which is determined by the numerical aperture of the focusing lens and the wavelength. In other words, substantial improvement of the spatial resolution is achieved. As discussed above, application of the double-resonance absorption process can realize fluorescence microscopy that overcomes the diffraction limit (super-resolution microscopy using the double-resonance absorption process).
In fluorescence microscopy, a specimen (mainly a biological specimen) usually is dyed with special molecules called "fluorescence probe" (or "fluorescence labeler"). A fluorescence probe absorbs a specific wavelength to emit relatively intense fluorescence. Application of such a fluorescence probe to the above-mentioned microscopy utilizing the double-resonance absorption process would be considered effective. However, only a few examples of molecules such as tryptophan and adenine have been applied to fluorescence microscopy in practice to prove their effectiveness as fluorescence probe. Tryptophan and adenine exist in the form of amino acid molecules or a base of a nucleic acid contained in biological cells. They are so special molecules as to emit fluorescence themselves in the cells.
In practice, kinds of molecules that can be observed without dye are limited in number; composition of most kinds of molecules cannot be known without dye. A fluorescence probe has a favorable property that a specific kind of molecules that are the object of observation can be selectively dyed with it upon its functional group been appropriately selected. To conform to the above-mentioned super-resolution microscope using the double-resonance absorption process, and to fully exploit its performance, it is necessary to select appropriate fluorescence probe molecules that could cause the above-mentioned double-resonance absorption process in an appropriate manner, as well as to optimize irradiation timing of the specimen with two wavelengths .lambda..sub.1 and .lambda..sub.2. Precedent studies, however, have neither disclosed an appropriate timing of irradiation nor specified certain molecules as the fluorescence probe.